Selective culture medium for microorganisms and use thereof



United States Patent 3,278,393 Patented Oct. 11, 1966 3,278,393SELECTIVE CULTURE MEDIUM FOR MICRO- ORGANISMS AND USE THEREOF Arthur N.Bahu, Evanston, and Irving L. Shklair, Wankegan, Ill., assignors toNorthwestern University, Evanston, 111., a corporation of Illinois NoDrawing. Filed May 1, 1964, Ser. No. 364,283 8 Claims. (Cl. 195-100)This invention relates to a selective culture medium for microorganisms.More particularly, this invention relates to a culture medium fordistinguishing Staphylococcus aureus and Pseudomonas aeruginosa fromother pathogenic microorganisms and others that may be morphologicallysimilar and from nonpatho-genic staphylococci organisms.

The staphylococci are by far the commonest cause of skin infections suchas boils, abcesses, carbuncles and similar suppurative processes in man.They are primarily significant as pathogens. They can be grown onculture media, such as agar or meat extract media, and the individualcolonies are circular with entire edges. The pathogenic forms, that is,those isolated from suppurative processes, usually are Staphylococcusaureus.

Staphylococci ferment mannitol with the formation of acid, the latterbeing detected by a decrease in pH of the media, as evidenced by colorchange of a pH indicator such as brom thymol blue or brom cresol purple.

Pathogenic staphylococci, that is, the Staphylococcus aurcus, causeclotting of decalcified blood plasma due to the production ofstaphylocoagulase, an active clotting agent. There is a high correlationbetween staphylococcal virulence for man and staphylocoagulaseproduction. Coagulase-positive bacteria are usually virulent.

Plasma clotting (i.e., staphylocoagulase activity) can be demonstratedby mixing a bacterial culture with decalcified plasma, human or rabbit,incubating the plasma and observing clotting. The simple slide method isoften used for qualitative purposes; the bacteria are suspended in adrop of plasma and observed on a slide for clumping.

Staphylococci constitute the normal flora of the human skin, mouth andupper respiratory tract and enter the body through the intact skin orbreaks therein. They include the relatively avirulent Staphylococcusepidermia'is, as well as the virulent Staphylococcus aureus, especiallyin the upper respiratory tract. The latter are the etiologic basis for avariety of skin lesions, as well as diseases of the respiratory tract.Staphylococcal infections have increased in prevalence and fatality inrecent years and are the predominant bacterial infections in thiscountry.

In the treatment of wounds and similar skin lesions it is oftendesirable, and sometimes necessary, to identify the microorganisms inthe wound for proper selection of antibiotic treatment. In the customarypractice, a culture is made of the wound or lesion in an agar medium andthe microorganisms developed therein. Staphylococcus aurcus is a commonpathogenic microorganism in skin wounds and it is desirable to be ableto identify these organisms promptly and accurately so that appropriatetreatment can be instituted without delay. For this reason it isdesirable to have a culture medium which contains an inhibitor whichinhibits the growth of most microorganisms except Staphylococcus aureusand thus allows rapid and accurate identification of Staphylococcusaureus if present in the wound or lesion.

Pseudomonas aeruginosa is a common cause of hospital infections,particularly in burns, and it is desirable to have a culture mediumwhich will aid in distinguishing this organism from other pathogens.

It is an object of this invention to provide a selective culture mediumfor microorganisms which inhibits the growth of nonpathogenic organisms,provides for the isolation of pathogenic staphylococci and distinguishespathogenic Staphylococcus aureus from other staphylococcal organisms. Itis another object to provide a selective culture medium which providesfor the isolation and identification of Pseudomonas aerugz'nosa. Theseand other objects will be apparent from and are achieved in accordancewith the following disclosure.

We have discovered that a culture medium containing a cadmium salt suchas cadmium nitrate, phosphate or chloride is effective as a selectivemedium for the culture of pathogenic Staphylococcus aureus. This culturemedium aids in the identification of pathogenic Staphylococcus aureusfrom other staphylococci and other microorganisms. The remainingcomponents in the culture medium are conventional materials such asagar, proteosepeptone fractions, potassium phosphate, yeast extract,peptones, mannitol and pH indicators which indicate acid production.

The isolation of Staphylococcus from a wound is usually not a difiicultmatter. Inoculation of a blood agar medium with the organism from apurulent specimen followed by 24 hours incubation at 37 C. gives a goodgrowth of colonies. With Staphylococcus aureus 24 hours incubation givesgood growth of creamy colonies that are surrounded by the clear zones of,8 hemolysis. When the medium is rendered selective by the inclusion ofthe cadmium salt, it is possible to isolate Staphylococcus aureus fromspecimens heavily contaminated with other bacteria.

As some Pseudomonas strains can grow in the cadmiuma-gar medium, it maybe necessary to distinguish them from Staphylococcus aureus. This can bedone by the mannitol fermentation and plasma coagulation tests. Themannitol fermentation test can be conducted in the same medium in whichthe bacteria are grown. A drop in pH of the medium indicates that themannitol has been converted to acid by fermentation. As Pseudomonasbacilli and nonpathogenic staphylococci (such as Staphylococcusepidermidis) do not ferment mannitol, they are readily distinguishedfrom Staphylococcus aureus. The plasma coagulation test confirms thisfact, as Pseudomonas bacilli and nonpathogenic staphylococci do notcoagulate decalcified blood plasma.

Another and prefered way of distinguishing Staphylococcus aureus fromPseudomonas strains, such as Pseudomortas aeruginosa, is by addition ofa soluble azide, such as sodium azide, to the culture medium. The azideinhibits pseudomonas strains while allowing the staphylococci todevelop.

Pseudomonas aeruginosa can be distinguished from Staphylococcus aureusby addition of a bile salt, such as sodium desoxycholate or othersoluble salt of desoxycholic acid, to the culture medium. Bile saltsinhibit the staphylococci without significantly affecting the growth ofPseudomonas strains.

The culture medium can be a standard agar medium containing 0.002 to 0.4gram of CdCl -2' A2H O per liter or equivalent amount of cadmium ascadmiun nitrate or cadmium phosphate. The culture medium can alsocontain D-mannitol, which has been used in other staphylooccus selectivemedia, in concentration in the range of 20 grams per liter to aid indistinguishing Staphylococcus aureus from pseudomonas and from otherstaphylococci organisms. A suitable c-admium-mannitol-agar culturemedium corresponds to the following formulation:

Formula in grams per liter of distilled water Proteose-peptone No. 3(Difco) Phytone (BBL) 10 K HPO 2.5 Yeast extract 2.5

D-mannitol 10 CdCl -2 /2H O 0.020 to 0.25 Agar An indicator such as0.04% brom thymol blue or 0.04% brom cresol purple may be added toindicate acid production by a color change.

The media of this specification can be used for the detection andenumeration of staphylococci. Most microorganisms with the exception ofPseudomonas are inhibited by the cadmium salt in the media. Theexception to this are fecal samples where Proteus may grow. Plates canbe heavily inoculated because of the inhibitory action of the cadmiumsalt. The inoculated plates are then incubated for 24-36 hours atapproximately 37 C. Most of the colonies that grow on the media willusually ferment the mannitol. Organisms which grow and do not fermentthe mannitol or coagulate plasma are nonpathogenic staphylococci orPseudomonas. Those that grow, ferment mannitol and coagulate plasma areStaphylococcus aureus pathogens.

Selective culture media for the detection and enumeration ofStaphylococcus aureus and for distinguishing it from Pseudomonasaeruginosa can be produced by the addition of 0.0004 to 0.4 gram perliter of sodium azide or other soluble azide to the media describedhereinabove. The azide will prevent growth of Pseudomonas strains, sothat the mannitol fermentation or plasma coagulation tests need not beused to distinguish Staphylococcus aureus from these strains. A typicalselective culture medium for Staphylococcus aureus is the following,which will give a pure culture in purulent wounds:

Formula in grams per liter of distilled water Proteose-peptone No. 3(Difco) 10 Phytone (BBL) 10 K HPO 2.5 Yeast extract 2.5 D-mannitol 10CdCl -2' /2H O 0.02 to 0.25 Agar 15 Sodium azide 0.1-0.2

Selective culture media for the detection of Pseudomonas aeruginosa andfor distinguishing this strain from staphylococci correspond to theforegoing formula with 0.005 to 10 grams per liter of a bile salt suchas potassium desoxycholate (or other soluble salt of desoxycholic acid)in lieu of the azide. A typical formula is the following:

Formula in grams per liter of distilled water Peptone 15 Phytone (BBL) 5Sodium citrate 15 Potassium gluconate 5 5 Glycerol 10 CdCl -2 /2H O 0.3Sodium desoxycholate 0.5

Media for isolation of Pseudomonas organisms can be incubated attemperatures from C. to 42 C. for periods of 24 to 48 hours. Media forisolation of staphylococci can be incubated at C. to 37.5 C. for periodsof 24 to 48 hours. Such media are aqueous media, whether in form ofbroths or agar gels, and are so described herein.

We claim:

1. A selective culture medium for the identification of microorganismswhich comprises an aqueous medium containing a cadmium salt selectedfrom the group consisting of cadmium nitrate, cadmium phosphate andcadmium chloride, the concentration of cadmium in said aqueous mediumbeing equivalent to 0.002 to 0.4 gram of CdCl -2' /2H O per liter, andD-mannitol, the concentration of the D-mannitol being in the range from5 to 20 grams per liter.

2. A selective culture medium for identification of microorganisms asdefined by claim 1 containing a soluble azide in concentration of 0.0004to 0.4 gram per liter.

3. A selective culture medium for identification of microorganisms asdefined by claim 1 containing a bile salt in concentration of 0.005 to10 grams per liter.

4. A selective culture medium for identification of microorganisms asdefined by claim 3 containing a soluble desoxycholate in concentrationof 0.005 to 10 grams per liter.

5. A method of identifying Staphylococcus aureus in a specimen ofmicroorganisms which comprises inoculating a culture medium with saidspecimen, the culture medium containing cadmium in an amount equivalentto 2 to 400 parts of CdCl -2 /2H O per million and containing 5 to 25grams D-mannitol per liter of culture medium, incubating the inoculatedmedium at approximately 37 C., measuring the change in pH of the medium,and removing from the culture medium a specimen of the colony growntherein and admixing said specimen with decalcified blood plasma,thereby identifying Staphylococcus aureus by its characteristics ofgrowing in a medium containing cadmium, fermenting D-mannitol to formacid, and coagulating decalcified blood plasma.

6. A method of isolating Staphylococcus aureus in a specimen ofmicroorganisms which comprises inoculating a culture medium with saidspecimen, the culture medium containing cadmium in an amount equivalentto 0.002 to 0.4 gram of CdCl -2V2H O per liter and containing a solubleazide in concentration of 0.0004 to 0.4 gram per liter, and incubatingthe medium at a temperature in the range of about 35 to 375 C., wherebythe Staphylococcus aureus is permitted to grow while other organisms areinhibited.

7. A method of isolating Pseudomonas aeruginosa in a specimen ofmicroorganisms which comprises inoculating a culture medium with saidspecimen, the culture medium containing cadmium in an amount equivalentto 0.002 to 0.4 gram of CdCl -2 /zH O per liter and containing a bilesalt in concentration of 0.005 to 10 grams per liter, and incubating themedium at a temperature in the range of about 20 to 42 C., whereby thePseudomonas aeruginosa is permitted to grow while other organisms areinhibited.

8. A method of identifying Staphylococcus aureus in a specimen ofmicroorganisms which comprises inoculating a culture medium with saidspecimen, the culture medium containing cadmium in an amount equivalentto 2 to 400 parts of CdCl '2 /2H O per million and containing 5 to 25grams D-mannitol per liter of culture medium, incubating the inoculatedmedium at approximately 37 C., measuring the change in PH of the medium,thereby identifying Staphylococcus aureus by its characteristics ofgrowing in a medium containing cadmium and fermenting D-mannitol to formacid.

References Cited by the Examiner Wallerstein LaboratoriesCommunications, vol. XIV, No. 47, pages 343 and 344 (1951).

A. LOUIS MONACELL, Primary Examiner.

ALV N E. TANENHOLTZ, Examiner.

8. A METHOD OF INDENTIFYING STAPHYLOCOCCUS AUREUS IN A SPECIMEN OFMICROORGANISMS WHICH COMPRISES INOCULATING A CULTURE MEDIUM WITH SAIDSPECIMEN, THE CULTURE MEDIUM CONTAINING CADMIUM IN AN AMOUNT EQUIVALENTTO 2 TO 400 PARTS OF CDCL2.C1/2H2O PER MILLION AND CONTAINING 2 TO 25GRAMS D-MANNITOL PER LITER OF CULTURE MEDIUM INCUBATING THE INOCULATEDMEDIUM AT APPROXIMATELY 37*C., MEASURING THE CHANGE IN PH OF THE MEDIUM,THEREBY IDENTIFYING STAPHYLOCOCCUS AUREUS BY ITS CHARACTERISTICS OFGROWING IN A MEDIUM CONTAINING CADMIUM AND FERMENTING D-MANNITOL TO FORMACID.